Abstract

We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca2+ channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure.

eLife digest

To hear a sound, the pressure produced by sound waves must be converted into an electrical nerve signal. The cells inside the ear that perform this transformation are called hair cells, which are so named because they have hundreds of hair-like structures on their upper surface. Pressure from sound waves causes movements in the inner ear that bend these ‘hairs’. This causes the hair cells to release chemical signals to neighboring nerve cell terminals that ultimately transmit information about the sound to the brain.

The chemical signals are stored inside the hair cells in bubble-like compartments called vesicles. To release the chemicals from the cell, the vesicles merge with the membrane that surrounds the hair cell. Most cells that communicate in this way are limited in how long they can transmit such messages. However, hair cells can continuously fuse vesicles to the membrane even when a sound lasts for a long time. This suggests that the hair cells have a different way of producing vesicles and getting them to the membrane than other cell types.

Inside the hair cells, vesicles are stored in regions called active zones. Each active zone contains a “ribbon” (attached to which are hundreds of vesicles) and also ion channels that allow calcium ions to flow into the cell. (An increase in calcium ion concentration inside the cell is necessary for the vesicle to fuse with the cell membrane and so release its chemical content). Now, Vincent et al. show that in hair cells, a cage-like network made from a protein called actin surrounds each active zone. This network helps to position the calcium ion channels. Treating the hair cells with a compound that disorganized the actin networks speed up the process of vesicle movement, which suggests that the actin network also controls the rate at which vesicles reach the membrane.

Next, it will be important to identify how the actin network interacts with other molecules that help vesicles to release their contents; in particular a protein called otoferlin, which is thought to act as a calcium ion sensor.

Confocal imaging of the synaptic F-actin cages in IHCs.

(A) Confocal images from averaged Z-stack projection (20 slices of 0.25 µm) of P13-IHCs labeled in blue with otoferlin-immuno-reactivity. Directly visualized with fluorescent-phalloidin (purple), F-actin intensively labelled the cuticular plate and the stereocilia but also in a punctated manner the synaptic basal pole of the IHCs. In this latter area, at higher magnification (averaged Z-stack projection of 8 slices of 0.25 µm), the synaptic F-actin forms a mesh of cages (see right panel where the blue channel of otoferlin is omitted; the cages are indicated by the white asterisks). At each border of the synaptic F-actin cages was generally attached one synaptic ribbon (red) and one associated Cav1.3 patch (green) as indicated in the lower left panel. The graph represents an example of fluorescent intensity profile through the white dashed line crossing the ribbon and the associated Cav1.3. (B) The graph indicates the Gaussian distribution of the larger axis (double white arrow head) of each F-actin cage. (C) A 45 min treatment with extracellular latrunculin-A disrupted the synaptic F-actin cages. The black holes at the base of the IHCs likely indicated swollen IHC active zones produced by the synaptic F-actin disorganization. At higher magnification (right panel), note also the disorganization of the Cav1.3 clusters (green) at the ribbons, as indicated by a larger distance in their respective fluorescent intensity profile distribution (bottom graph). (D) Comparative Gaussian distribution of the center mass distance between Cav1.3 and ribbon in controls (black, n = 71 active zones) and latrunculin-treated (orange, n = 102 active zones) IHCs. The inset histogram indicates the mean ± SEM distance in both conditions. *p<0.05.

A 45-min treatment of the organ of Corti in vitro with 1 µM extracellular latrunculin-A completely disorganized the synaptic F-actin cages and the Cav1.3 immunoreactive-patches at the IHC ribbons (Figure 1C, D). The mean distance between the Cav1.3 immunoreactive patches and the ribbons increased from 218 ± 11 nm (n = 71) in controls to 260 ± 12 nm with latrunculin-A treatment (n = 102; p<0.05). In the latter conditions, surprisingly, whole-cell patch clamp recordings revealed a largely facilitated exocytosis as compared to controls, while voltage-gated Ca2+ currents were unchanged (Figure 2A,B). After 100 ms depolarization, from -80 to -10 mV, the exocytotic response in control IHCs reached a maximum amplitude of 22.0 ± 2.9 fF (n = 11). Considering that a 40 nm diameter synaptic vesicle corresponds to 37 aF (Lenzi et al., 1999), we estimated a RRP size of about 590 vesicles, i.e 33 vesicles per ribbons if we assume a number of 18 ribbons per IHCs. This number of RRP vesicles fit well with previous findings (Johnson et al., 2008; Vincent et al., 2014). In latrunculin-treated IHCs, the exocytotic responses reached, at 100 ms, 32.4 ± 3.3 fF (n = 18), a value significantly larger as compared to control IHCs (p<0.05; Figure 2B). Remarkably, while the exocytotic response saturated at 90–100 ms in controls, the response did not show saturation in latrunculin-treated IHCs. Since the exocytotic response was unchanged for short impulses below 30 ms (Figure 2B), these results suggested that the disruption of F-actin did not affect the steps of vesicle docking and priming but facilitated the replenishment of the RRP. In bassoon mutants with abnormal number of anchored ribbons and reduced Ca2+ currents both short (20 ms) and long (100 ms) impulse activated-exocytosis were affected (Jing et al., 2013).

Latrunculin-A treatment facilitated exocytosis in IHCs.

(A) Ca2+ currents, evoked by a voltage-ramp protocol, were not significantly affected by latrunculin-A (orange). The parameters of the Boltzman fit are indicated by the histograms (mean ± SEM). Rising intracellular osmotic pressure from 310 mOsm to 390 mOsm did not affect the Ca2+ currents in latrunculin-treated IHCs (pink). For each curves, the darker line indicates the mean responses and the light filled area the standard error. (B) Exocytosis evoked by voltage-steps from -80 mV to -10 mV was largely facilitated after latrunculin-A treatment. At right, examples of Ca2+ currents and capacitance jumps (△Cm) are shown for a control (black traces) and latruncutin-treated IHC (orange traces). In 5 mM intracellular EGTA condition, the facilitation was greatly reduced (blue points). Data are represented as mean ± SEM. *p<0.05. (C) Exocytosis under Ca2+ uncaging was also facilitated after latrunculin-A treatment. The jump in the concentration of intracellular free Ca2+ was similar in control and latrunculin-treated IHCs (inset; p = 0.9). (D) Left, comparative exocytotic rates of control and latrunculin-treated IHCs obtained from the first derivative (dCm/dt) of the curves in C. Right, comparative maximum exocytotic amplitude and peak rate histograms (mean ± SEM). Increasing intracellular hydrostatic pressure from 310 to 390 mOsm (pink bars) did not affect exocytosis in latrunculin-treated IHCs. Numbers of cells are indicated in the histogram. *p<0.05 and n.s. as non significative.

The exocytotic facilitation in latrunculin-treated IHCs was greatly reduced to 21.8 ± 2.5 fF at 100 ms (n = 7) and comparable to controls (p=0.9) when intracellular Ca2+ buffering was increased with 5 mM EGTA (Figure 2B). This sensitivity to EGTA suggested a spatial disorganization of the Ca2+ channel clusters in regards to the release sites in latrunculin-treated IHCs, in good agreement with confocal imaging (Figure 1C,D). Rising the EGTA concentration could also affect the extent to which neighboring calcium sources interact and summate to produce a global effect on free calcium.

The intriguing question now is: why is the disruption of the synaptic F-actin with latrunculin-A facilitating exocytosis in IHCs? One explanation is that the synaptic F-actin network, in addition to organizing Ca2+ microdomains, also acts as a diffusion barrier for synaptic vesicles limiting the access to the site of release, as suggested in some central synapses (Cingolani and Goda, 2008). Its disruption with depolymerising agents would therefore facilitate vesicle replenishment of the release sites, as shown in a large variety of secretory cells (Malacombe et al., 2006), by increasing the number of available vesicles for docking and priming. Alternatively, a disrupted F-actin could facilitate the diffusion of Ca2+ from its sites of entry and stimulate replenishment.

To directly test these hypothesis, we studied the effect of F-actin depolimerization on exocytosis triggered by intracellular Ca2+ uncaging, i.e. independently of the activation and the organization of the Ca2+ channels. In these experiments, a large exocytotic facilitation was also observed in latrunculin-treated IHCs (Figure 2C,D). The peak exocytotic rate, obtained by measuring the first derivative function of the curves in Figure 2C, was nearly two fold larger in latrunculin-treated IHCs as compared to controls (Figure 2D; 25.9 ± 2.7 pF/s (n = 14) and 13.7 ± 1.7 pF/s (n = 16), respectively; p<0.05). The levels of the peak intracellular Ca2+ concentration ([Ca2+]free) reached upon UV-flash Ca2+ uncaging were verified to be similar in control (n = 7) and latrunculin-treated IHCs (n = 5), respectively 59 ± 7 µM and 57 ± 5 µM (p=0.9; Figure 2C-inset). These Ca2+ uncaging experiments again suggested that a synaptic F-actin network controls the diffusion rate of the synaptic vesicles to the sites of release in IHCs.

Furthermore, since intracellular hydrostatic pressure has been suggested to influence membrane tension and exocytosis through the F-actin network in many cell types such as mast cells (Solsona et al., 1998), we probed Ca2+-evoked exocytosis under various intracellular osmotic pressures in auditory IHCs. We first found that increasing intracellular osmotic pressure from 310 to 390 mOsm with sucrose produced a significant increase in the resting membrane capacitance of IHCs. The resting size of IHCs, voltage-clamped at -80 mV for a period of 2 min after break-in, was 9.93 ± 0.27 pF (n = 11, 310 mOsm) and 11.10 ± 0.34 pF (n = 10, 390 mOsm; p<0.05), respectively. This augmentation of the IHC resting membrane capacitance was about 50 times larger than the size of the RRP evoked by membrane depolarization (Figure 2B; RRP = 22 fF). Where does this large addition of membrane come from? One possible explanation was that high intracellular hydrostatic pressure triggers the fusion of a large amount of extrasynaptic vesicles to the plasma membrane, as previously suggested for Ca2+ uncaging (Vincent et al., 2014).

Remarkably, in these latter conditions of intracellular hyperosmotic stress at 390 mOsm, voltage-dependent Ca2+ currents displayed larger amplitude (Figure 3A,C) and accelerated activation kinetics as compared to control conditions at 310 mOsm (Figure 3E). A shift in their voltage-dependence toward more negative potentials was also observed (Figure 3A,D). The hydrostatic pressure effects on Ca2+ currents were no longer visible when IHCs were pre-treated with latrunculin-A (Figure 2A, pink line) and were greatly reduced in IHCs lacking otoferlin (Otof-/-; Figure 3B,C). This indicated that the Cav1.3 channels of IHCs are mechanosensitive, like the Cav1.2 channels in smooth muscle cells (Lyford et al., 2002). We cannot exclude the addition of Ca2+ channels to the plasma membrane during the massive vesicular fusion caused by increased hydrostatic pressure. However, this mechanism appears unlikely in regards to results obtained in non-secretory cells such as smooth muscle and HEK cells where similar effect on Ca2+ channels were obtained (Lyford et al., 2002). In our study, the sensitivity to membrane tension in IHCs required an intact synaptic F-actin network and otoferlin, a proposed synaptic Ca2+ sensor thought to interact physically with the Cav1.3 II-III loop (Ramakrishnan et al., 2009). Notably, an increase in Ca2+ current amplitude in low bath hydrostatic pressure (equivalent to increasing intracellular pressure) was also reported in dissociated guinea pig vestibular hair cells (Duong Dinh et al., 2009; Haasler et al., 2009). In these latter studies, the change in Ca2+ currents could be interpreted as due to a pressure activation of K+ currents leading to less pronounced depolarization. Although pressure effects on K+ currents have also been shown in guinea-pig IHCs (Kimitsuki, 2013), we don't think that these currents would influence our Ca2+ current measurements since we were working in conditions where most K+ currents are blocked. Overall, our results suggested here that the mechanosensitivity of the Cav1.3 channels is mediated through an intact synaptic F-actin network.

(A) Comparative Ca2+ currents evoked by a voltage-ramp protocol in Otof+/- and Otof+/+IHCs (expressing otoferlin). Cells were recorded with intracellular osmotic pressure at 310 mOsm (n = 10) or at 390 mOsm (n = 9). The darker line indicates the mean responses and the light filled area the standard error. (B) Ca2+ currents in Otof-/- IHCs (lacking otoferlin) recorded in conditions similar to A. (C-D) Both maximum amplitude and half-maximum voltage-activation of Ca2+ currents were maximally affected at 390 mOsm. Note that the slopes of the Boltzman fit of the activation curve in A and B were found slightly affected in IHCs expressing otoferlin (310 mOsm: K = 5.90 ± 0.14 pA/mV, n = 10 and 390 mOsm: K = 5.36 ± 0,09 pA/mV, n = 9; p < 0.05) but not in Otof-/-IHCs (310 mOsm: K= 6.18 ± 0.20 pA/mV, n=6 and 390 mOsm: K= 5.89 ± 0.28, n = 6; p = 0.71) (data not shown). Data are represented as mean ± SEM, with the number of cells indicated above each point and (*) indicating p < 0.05. (E) Activation kinetics of Ca2+ currents, evoked by voltage-steps from -80 mV to different voltage levels, were significantly faster at 390 mOsm in hair cells expressing otoferlin but not in Otof-/- IHCs. For unknown reason the activation kinetics were faster in Otof-/-IHCs as compared to IHCs expressing otoferlin. Data are expressed as mean ± SEM. * p < 0.05. The number of cell is similar to D. (F) Ca2+ efficiency of exocytosis was plotted as the change in membrane capacitance (ΔCm) against the integral of the calcium current (QCa) when depolarizing IHCs at a constant voltage-step from -80 to -10 mV with increasing duration from 10 to 120 ms. Data points at 310 mOsm and 390 mOsm were fitted with a power function with a Ca2+ efficiency slope A = 2.47 ± 0.45 fF/pC and 9.30 ± 2.5 fF/pC (p < 0.05) and a (power cooperative index) = 0.33 and 0.51 (p = 0.52), respectively. The supralinear power cooperative index of 0.3 and 0.51 found here was somewhat lower to previous values reported by Cho et al. (2011) and Johnson et al (2005). (G) The exocytotic efficiency of each data point in F (ΔCm/QCa) was plotted for each depolarizing time at 310 mOsm and 390 mOsm. Data were fitted with an exponential function with 22.2 ± 10.4 ms and 50.1 ± 11.9 ms, respectively (p < 0.05). (H) Ca2+ efficiency of exocytosis (recorded in conditions similar to F) was unaffected when rising osmotic pressure from 310 to 390 mOsm in Otof-/-IHCs. Data were best fitted with a linear function with a similar slope of 0.12 ± 0.1 fF/pC and 0.48 ± 0.1 fF/pC (p = 0.2) at 310 and 390 mOsm, respectively. Data are represented as mean ± SEM. * p < 0.05.

Exocytosis triggered by voltage-activation of Ca2+ channels from -80 to -10 mV (a voltage at which Ca2+ current amplitude is maximum, Figure 3A) also showed maximum facilitation when intracellular pressure was increased from 310 to 390 mOsm (Figure 3F,G). Notably, on the contrary to what observed when disrupting the synaptic F-actin, exocytotis increased for short impulses (10–20 ms) at 390 mOsm (Figure 3F,G), suggesting that the last steps of vesicle exocytosis (docking, priming and/or fusion) were here accelerated. Exocytosis for longer steps up to 90 ms also increased, from 1.40 ± 0.23 fF/pC (n = 7, 310 mOsm) to 2.58 ± 0.49 fF/pC (n = 5, 390 mOsm; p<0.05) at 50 ms (Figure 3G). These latter results suggested that the replenishment rate of the RRP was also accelerated.

This facilitation of exocytosis, under intracellular hyperosmotic stress, appeared unrelated to the effects on Ca2+ channels since it was also observed when directly uncaging intracellular Ca2+ (Figure 4). The levels of the peak [Ca2+]free reached upon UV-flash Ca2+ uncaging were verified to be similar in 310 mOsm (n = 7) and 390 mOsm (n = 5) conditions, 59 ± 7 µM and 64 ± 6 µM (p=0.6; Figure 4A-inset). In these latter conditions of intracellular hyperosmotic stress, the strength of exocytotic facilitation was maximal at 390 mOsm and abruptly decreased at 410 mOsm, a high pressure at which the intrinsic organization of the IHC exocytotic machinery may have been damaged. At 390 mOsm, the maximum amplitude of the exocytotic response was 1.5 ± 0.15 pF (n = 9) as compared to 1.0 ± 0.1 pF (n = 16) in controls at 310 mOsm (p<0.05; Figure 4A,C). The peak exocytotic rate was largely increased at 390 mOsm as compared to 310 mOsm (25.0 ± 4.5 pF/s (n = 9) and 13.7 ± 1.7 pF/s (n = 16), respectively; p<0.05, Figure 4D). Interestingly, the facilitation of exocytosis by high intracellular hydrostatic pressure was not observed in IHCs treated with latrunculin-A (Figure 2D, pink bars) and in Otof-/-IHCs (Figure 3H and Figure 4B–D). The peak [Ca2+]free reached upon UV-flash Ca2+ uncaging was unchanged in Otof-/-IHCs as compared to control IHCs (Vincent et al., 2014).

(A) Exocytosis in IHCs expressing otoferlin was largely potentiated when rising osmotic pressure from 310 mOsm (light grey) to 390 mOsm (dark grey). The darker line in each condition indicates the mean responses and light filled area the standard error. The jump in the concentration of intracellular free Ca2+ was similar in 310 and 390 mOsm conditions (inset; p = 0.6). (B) Exocytosis evoked in Otof-/- IHCs in conditions similar to A. (C-D) Comparative maximum amplitude and peak exocytotic rate at various intracellular osmotic pressure. The results showed a maximum facilitation at 390 mOsm. These pressure effects were not seen on the residual slow exocytosis of Otof -/- IHCs. Data points are means ± SEM. The number of IHCs is indicated above each point. * p < 0.05.

Overall, the facilitation of exocytosis by high intracellular hydrostatic pressure could be explained by an increased membrane tension that impacts on membrane fusion (Kozlov and Chernomordik, 2015) but also by an increased vesicular mobility, possibly as the result of reduced molecular crowding and loosened vesicle interactions, accelerating the replenishment rate. In the same way, the mobility of vesicles in pancreatic cells and primary hepatocytes was shown to be affected by hydrostatic pressure, a process related to molecular crowding and microfilaments polymerization (Nunes et al., 2015).

Do large changes in hydrostatic pressure occur in the cochlea during physiological or pathological conditions? In the cochlea, large intercompartmental osmotic gradients from 289 to 322 mOsmol/kg H20 are present between the perilymphatic and endolymphatic compartments, respectively (Sterkers et al., 1984). These osmotic gradients are likely regulated by aquaporins present in outer sulcus cells (Eckhard et al., 2015) and genetic deletion of aquaporin-4 in mice leads to impaired hearing (Li and Verkman, 2001). Osmolarity changes in these inner fluid compartments have long been suspected to be contributing factors to inner ear disorders such as tinnitus and fluctuating hearing loss, inclusive of Menière's disease (Angelborg et al., 1982). The outer hair cells, producing the electro-mechanical amplification of sound in the cochlea, have been shown to be mechanically sensitive to extracellular osmotic variation (Dulon, et al., 1987), a factor that greatly influence hearing (Choi and Oghalai, 2008). Our study, showing that IHC exocytosis is sensitive to osmotic forces, suggests that a defect in synaptic transmission at the auditory ribbon synapses may also underlie some hearing disorders associated with Menière's disease.

In conclusion, we propose that a synaptic F-actin network tightly controls the flow of synaptic vesicles during exocytosis at the IHC ribbons. This synaptic F-actin network also influences the sensitivity of exocytosis to hydrostatic pressure changes, presumably through a regulation of membrane tension at the active zones. Such actin-based regulation of intracellular hydrostatic pressure and membrane tension, recently shown in a variety of physiological processes such as in the secretory properties of neuroendocrine cells (de Wit, 2010; Gutiérrez and Gil, 2011), the mitotic cell rounding during cell division (Stewart et al., 2011), the photomechanical responses of photoreceptors (Hardie and Franze, 2012) to name a few, is here described for the first time in auditory hair cells.

Materials and methods

Tissue preparation

Experiments were performed in accordance with the guidelines of the Animal Care Committee of the European Communities Council Directive (86/609/EEC) and were approved by the ethics committee of the University of Bordeaux (animal facility agreement number C33-063-075). All mice (C57BL6 of either sex) were anesthetized by intraperitoneal injection of xylazin (6 mg/ml) and ketamine (80 mg/ml) mixture (Sigma Aldrich, St Louis, USA) diluted in physiological saline. The organs of Corti were dissected as previously described (Vincent et al., 2014).

Electrophysiological recordings were obtained from littermate Otof+/+, Otof+/- or from knock-out (KO) otoferlin (Otof-/-) C57BL6 mice at postnatal day 12–16 (P12-P16) inner hair cells (IHCs) in whole-mount organs of Corti in the apical cochlear area coding for frequencies ranging from 8 to 16 kHz.

Current-voltage (I-V) curves were recorded using two different protocols. First, cells were maintained at -80 mV and depolarized with a ramp protocol (1 mV/ms) from -90 mV to +30 mV in 120 ms. The voltage parameters (half max activation potential and the slope) of the IV curves were given by fitting the IV curves from -70 mV to -10 mV with a Boltzmann sigmoidal function:

(1)y=A1−A21+exp(V−V1/2)/K

Where A1 and A2 are the minimum and the maximum y value. V is the voltage value and V1/2 is the half max voltage activation, K is the slope of the IV curve. Second, cells were step-depolarized from -80 mV to -5mV in 5mV increments for a constant time duration of 100 ms. Activation kinetics of the Ca2+ currents were determined at different potentials with the latter protocol by using a single exponential fit:

(2)y=y0+A1exp(−x/t)

Where yois the offset of activation, A1 the amplitude of the Ca2+ current and t the time constant.

Real-time capacitance measurements (Cm) were performed using the Lock-in amplifier Patchmaster software (HEKA) by applying a 1 kHz command sine wave (amplitude 20 mV) at holding potential (-80 mV) before and after the pulse experiment, as previously described (Vincent et al., 2014). The time interval between each depolarization was set at 10 seconds to allow full replenishment of the RRP. The exocytosis efficiency was determined by fitting the data points with a power function:

(3)ΔCm=y0+A[x−xc]a

Where yois the initial value (fF), A the Ca2+ efficiency slope (fF/pC), a the power cooperative index, xc the Ca2+ charge (QCa in pC) threshold of the response and x (pC) the QCa value for each stimulation. The relationship between the Ca2+ efficiency and the time was fitted with a exponential function:

(4)y=y0+A1e−(x−x0)/t

Where yoet x0 are the y and x offset, A1 the amplitude and t the time constant.

Only cells with stable membrane resistance (Rm), leak current below 50 pA at holding potential (-80 mV) and stable series resistance below 15 MΩ were considered in the study. All Ca2+ currents were leak-subtracted.

Caged Ca2+ photolysis

To trigger a fast increase in intracellular Ca2+ concentration from the caged Ca2+ chelator DM-nitrophen (Interchim, France), we used 200 ms brief flashes from a UV LED light source (Mic-LED 365, 128mW, Prizmatix, Givat Shmuel, Israel). The UV LED was directly connected to the epi-illumination port at the rear of our upright Nikon FN1 microscope and illumination was focalized through the 60X objective (CFI Fluor 60X W NIR, WD = 2.0 mm, NA=1). Hair cells were loaded with in mM, CsCl 145; HEPES 5; TEA 20; DM-nitrophen 10; CaCl2 10. In some experiments, in order to check that similar Ca2+ release occurs in all our conditions, we added 50 µM OGB-5N in the intracellular solution. To determine the intracellular Ca2+ concentration ([Ca2+]free), we used the following formula:

(5)[Ca2+]free=KD×(F−Fmin)(Fmax−F)

Where [Ca2+] free is the caged Ca2+ released by the UV flash, KD the constant dissociation of OGB-5N. We used the value of 23.3 µM determined by our previous in vivo calibration (Vincent et al., 2014). F the over time fluorescence, Fmin the minimum fluorescence and Fmax the maximal fluorescence. Fluorescence signals were plotted and compared as ΔF/Fmin where ΔF is equal to: F-Fmin. To vary the intracellular osmotic pressure from 310 to 360, 390 or 410 mOsm, the DM-nitrophen solution was complemented with sucrose as described above.

Confocal imaging was performed with a Leica SP8 confocal laser-scanning microscope with a 63X oil immersion objective (NA = 1.4) and white light laser (470 to 670 nm) (Bordeaux Imaging Center). Phalloidin was imaged by using a diode laser at 405 nm also mounted on the microscope.

Statistical analysis

Electrophysiological results were analyzed with Patchmaster (HEKA Elektronik, Germany), OriginPRO 9.1 (OriginLab, Northampton, USA) and IgorPro 6.3 (Wavemetrics, Oregon, USA). Results are expressed as mean ± SEM. Statistical analyses were performed by using the non-parametric Mann-Whitney U test with OriginPRO 9.1 software. The number of samples for each condition was indicated on graphs or in legends. The limit of significance was set at 0.05 (p<0.05). When the statistical tests were found to be non-significant, the p value was given.

Decision letter

Christian Rosenmund

Reviewing Editor; Charité, Universitätsmedizin Berlin, Germany

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled "A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission. The reviewers thought that the work contains interesting new data that will be of interest to a wide audience of neuroscientists and cell biologists that justify publication in eLife. No new experiments are required, but we ask you to address their concerns related to experimental conditions (osmolarity), text editing and data analysis, which you will find below in the original comments.

Reviewer #1:

This is a very interesting paper with some surprising results. The role of actin in the process of exocytosis is unknown for auditory hair cell ribbon synapses, although these synapses have an abundant supply of actin, as shown in Figure 1 of this paper. At other synapses, it is thought that actin modulates the process of exocytosis and the recruitment of vesicles to the readily releasable pool (RRP). The authors of this paper show that a 45-minute treatment with latrunculin-A, a drug that depolymerizes actin, increases the amount of exocytosis in hair cells. Interestingly, it also appears to allow Ca2+ channels to be located further away from the synaptic ribbon. It thus appears that actin is involved in stabilizing the exact localization of Ca2+ channels at synaptic ribbon active zones. These are important and novel findings. They will be of interest to a wide audience of neuroscience and cell biology. I have only a few major concerns that need to be addressed:

1) In Figure 2C the authors should state whether the level of peak free Ca2+ concentration reached in both condition (control and latrunculin-A treated) was the same. If available, please show the Ca2+ concentration levels in panel 2C.

2) Another way to quantify the inflexion point (or breaking-point) is to calculate the first derivative of the traces in Figure 2C and then show a bar graph of the peak values of the first derivative. Is this what the authors have done? If not, I would like to see this also shown in panel 2D.

3) Changing the osmolarity of the internal solution 310 to 390 mOsm seems like a drastic change. Does this ever occur under physiological or pathological conditions? Please discuss this further.

4) Is there some particular physiological context where an osmolarity change from 310 to 390 can occur in the mammalian organ of Corti? Perhaps under pathological conditions? The authors need to discuss this more fully.

5) The increase in capacitance jumps with latrunculin-A seems to suggest that actin acts as a barrier that controls the trafficking of vesicles from the cytoplasm to the release sites near the synaptic ribbon. Electron-tomography evidence for actin and microtubules near the synaptic ribbon has been provided by Graydon et al. (JNeurosci., 2011; see their Figure 5B). This paper should be cited in the Introduction.

6) The level of free Ca2+ concentration reached in Figure 4A and 4B should be mentioned in the text or the figure caption. Average numbers should be given in the text for this set of experiments.

In the paper the authors present a fascinating series of observations linking an intracellular actin network to trafficking of synaptic vesicles, and potentially to changes in hydrostatic pressure. F-actin 'cages' appear to surround hair cell active zones. When cages are disrupted by latrunculin, the spacing of synaptic ribbons and calcium channel clusters is widened. Latrunculin treatment increases the voltage-evoked capacitance increase thought to reflect fusion of synaptic vesicles. Increased hydrostatic pressure also increased voltage-gated calcium current. Calcium-invoked capacitance increases are enhanced by increased hydrostatic pressure. This work raises many interesting questions. The initial observation of F-actin-dependent synaptic structure and function is significant and potentially quite important, particularly in view of the organized membranous domains of hair cells (see for example, Bullen et al., J Cell Sci. 2015 Jul 15;128(14):2529-40). A number of suggestions and questions follow:

1) Introduction, first paragraph: Introducing hair cells as 'highly sensitive barometers […]' in a paper that examines the effects of hydrostatic pressure might mislead many readers to think that the authors are suggesting that hair cells are stimulated by changes in hydrostatic pressure at acoustic frequencies. But of course the adequate stimulus is hair bundle displacement, not cytoplasmic pressure.

2) Results and Discussion, first paragraph – cages or cylinders? It’s not obvious from the images. Are these maximum intensity z-stack projections? We need more information in the figure legends.

4) Results and Discussion, second paragraph: Can the authors provide an estimate of the number of vesicles per ribbon that make up the RRP? Does it make sense?

5) Results and Discussion, second paragraph: "[…] a switch from nanodomain to microdomain […]." This seems like an unwarranted throw away statement in this context. The capacitance change being measured occurs over 100 ms, effectively steady-state with respect to buffer effects. EGTA concentration could affect the extent to which neighboring calcium sources interact and summate to produce a global effect on free calcium – is that what the authors intend? Without further explanation this reference to nano/micro is irrelevant.

6) Results and Discussion, second paragraph: The authors refer here to "number of docked vesicles at release sites", but later to "diffusion rate of the synaptic vesicles to the sites of release". These are quite different mechanisms to explain the effect of latrunculin, with the second idea probably closer to reality. What is the authors' conclusion?

7) Results and Discussion, third paragraph: ~1.2 pF capacitance increase with increased pressure, approximately equivalent to the calcium-evoked capacitance increase in control and latrunculin-treated cells (0.9 to 1.4 pF), but – 30-40-fold greater than RRP. So presumably not just synaptic vesicles – what other organelles could be involved in these much larger membrane fusions?

8) Results and Discussion, third paragraph: could the authors clarify the reference to 'mechanosensitive' Cav1.3 channels. Is it just that calcium channels are added to the membrane during the massive vesicular fusion caused by increased hydrostatic pressure?

9) In the Results and Discussion, third paragraph, please rephrase the following passage: “Notably, a change in Ca2+ current amplitude…”. The authors should replace “change” for “reduction”.

10) Results and Discussion, last paragraph: F-actin regulates vesicle trafficking by sensing and regulating focal changes in hydrostatic pressure? What are these 'focal changes in hydrostatic pressure'? How would these be generated?

11) Figure 1: provide hair cell outline in right hand panel at hi mag. Is this a z-axis projection? Also, panel B is mislabeled as panel C on figure. What are the large black holes at the bottom of latrunculin-treated IHCs? What are the dimensions of F-actin cages in latrunculin? Are these center-to-center distances of ribbon and CaV1.3? Please specify in the figure legend.

Author response

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission. The reviewers thought that the work contains interesting new data that will be of interest to a wide audience of neuroscientists and cell biologists that justify publication in eLife. No new experiments are required, but we ask you to address their concerns related to experimental conditions (osmolarity), text editing and data analysis, which you will find below in the original comments.

Reviewer #1:

[…] I have only a few major concerns that need to be addressed: 1) In Figure 2C the authors should state whether the level of peak free Ca2+ concentration reached in both condition (control and latrunculin-A treated) was the same. If available, please show the Ca2+ concentration levels in panel 2C.

The levels of the peak intracellular Ca2+ concentration ([Ca2+]free) reached upon UV-flash Ca2+ uncaging were verified to be similar in control (n = 7) and latrunculin-treated IHCs (n = 5), respectively 59 ± 7 µM and 57 ± 5 µM (p = 0.9). These data are now added in Figure 2C (inset) and discussed in the fifth paragraph of the Results section.

2) Another way to quantify the inflexion point (or breaking-point) is to calculate the first derivative of the traces in Figure 2C and then show a bar graph of the peak values of the first derivative. Is this what the authors have done? If not, I would like to see this also shown in panel 2D.

As suggested by the reviewer, we analyzed the first derivative of the exocytotic curve of Figure 2C and plotted the results in Figure 2D. Peak rate values, instead of breaking points, are now indicated as mean ± SEM in a bar graph (Figure 2D, bottom right). Note that the analysis of additional cells bring the n values from 11 to 16 in controls and from 10 to 14 for latrunculin-A treated IHCs. This new data analysis is now discussed in the fifth paragraph of the Results section.

3) Changing the osmolarity of the internal solution 310 to 390 mOsm seems like a drastic change. Does this ever occur under physiological or pathological conditions? Please discuss this further.

4) Is there some particular physiological context where an osmolarity change from 310 to 390 can occur in the mammalian organ of Corti? Perhaps under pathological conditions? The authors need to discuss this more fully.

A discussion on the points raised by the reviewer is now in the eleventh paragraph of the Results section: ‘Do large changes in hydrostatic pressure occur in the cochlea during physiological or pathological conditions? […] Our study, showing that IHC exocytosis is sensitive to osmotic forces, suggests that defects in synaptic transmission at the auditory ribbon synapses may also underlie some hearing disorders associated with Menière's disease.’

5) The increase in capacitance jumps with latrunculin -A seems to suggest that actin acts as a barrier that controls the trafficking of vesicles from the cytoplasm to the release sites near the synaptic ribbon. Electron-tomography evidence for actin and microtubules near the synaptic ribbon has been provided by Graydon et al. (JNeurosci., 2011; see their Figure 5B). This paper should be cited in the Introduction.

This point has been added in the Introduction: ‘Electron-tomography evidence for F-actin and microtubules near the synaptic ribbon has been observed in bullfrog hair cells (Graydon et al 2011).’

6) The level of free Ca2+ concentration reached in Figure 4A and 4B should be mentioned in the text or the figure caption. Average numbers should be given in the text for this set of experiments.

This point has been added: ‘The level of peak [Ca2+]int reached upon UV-flash Ca2+ uncaging were verified to be similar in 310 mOsm/kg (n=7) and 390 mOsm/kg (n = 5) conditions, 59 ± 7 µM and 64 ± 6 µM (p = 0.6)’. These data are now added in Figure 4A (inset) and discussed in the ninth paragraph of the Results section.

This recent paper is now discussed. It is now indicated in the tenth paragraph of the Results section: ‘In the same way, the mobility of vesicles in pancreatic cells and primary hepatocytes was shown to be affected by hydrostatic pressure, a process related to molecular crowding and microfilaments polymerization (Nunes et al., 2015).’

Reviewer #2:

[…] A number of suggestions and questions follow:

1) Introduction, first paragraph: Introducing hair cells as 'highly sensitive barometers […]' in a paper that examines the effects of hydrostatic pressure might mislead many readers to think that the authors are suggesting that hair cells are stimulated by changes in hydrostatic pressure at acoustic frequencies. But of course the adequate stimulus is hair bundle displacement, not cytoplasmic pressure.

This sentence has been changed in the Introduction and now reads: ‘Auditory hair cells convert tiny variations of sound pressure through the displacement of their apical hair bundles into analogous voltage waveforms.’

2) Results and Discussion, first paragraph – cages or cylinders? It’s not obvious from the images. Are these maximum intensity z-stack projections? We need more information in the figure legends.

The term cylinder is confusing and replaced by cage in the first paragraph of the Results and Discussion section. The confocal images shown in Figure 1 are averaged Z-stack projections of 20 (top left) to 8 (top right and bottom left) slices of 0.250 µm. By varying the depth of the Z-axis, a F-actin cage-like structure was observed surrounding the ribbon. These points are now added in the legend of Figure 1.

4) Results and Discussion, second paragraph: Can the authors provide an estimate of the number of vesicles per ribbon that make up the RRP? Does it make sense?

We discussed this point in the second paragraph of the Results section (please see: ‘After 100 ms depolarization, from -80 to -10 mV, the exocytotic response in control IHCs reached a maximum amplitude of 22.0 ± 2.9 fF (n = 11). […] In bassoon mutants with abnormal number of anchored ribbons and reduced Ca2+ currents both short (20 ms) and long (100 ms) impulse activated-exocytosis were affected (Jing et al., 2013)’).

5) Results and Discussion, second paragraph: "[…] a switch from nanodomain to microdomain […]." This seems like an unwarranted throw away statement in this context. The capacitance change being measured occurs over 100 ms, effectively steady-state with respect to buffer effects. EGTA concentration could affect the extent to which neighboring calcium sources interact and summate to produce a global effect on free calcium – is that what the authors intend? Without further explanation this reference to nano/micro is irrelevant.

We agree with the reviewer. This section has been now modified to read: ‘This sensitivity to EGTA suggested a disorganization of the Ca2+ channel clusters in regards to the release sites in latrunculin-treated IHCs, in good agreement with confocal imaging (Figure 1C, 1D). Rising the EGTA concentration could also affect the extent to which neighboring calcium sources interact and summate to produce a global effect on free calcium.’

6) Results and Discussion, second paragraph: The authors refer here to "number of docked vesicles at release sites", but later to "diffusion rate of the synaptic vesicles to the sites of release". These are quite different mechanisms to explain the effect of latrunculin, with the second idea probably closer to reality. What is the authors' conclusion?

The sentence has been modified (now in the Results and Discussion, fourth paragraph) to read: ‘Its disruption with depolymerising agents would therefore facilitate vesicle replenishment of the release sites, as shown in a large variety of secretory cells (Malacombe et al., 2006), by increasing the number of available vesicles for docking and priming. Alternatively, a disrupted F-actin could facilitate the diffusion of Ca2+ from its sites of entry and stimulate replenishment.’

7) Results and Discussion, third paragraph: ~1.2 pF capacitance increase with increased pressure, approximately equivalent to the calcium-evoked capacitance increase in control and latrunculin-treated cells (0.9 to 1.4 pF), but – 30-40-fold greater than RRP. So presumably not just synaptic vesicles – what other organelles could be involved in these much larger membrane fusions?

This point is now discussed in the sixth paragraph of the Results and Discussion section as follows: ‘This augmentation of the IHC resting membrane capacitance was about 50 times larger than the size of the RRP evoked by membrane depolarization (Figure 2B; 22 fF). Where does this large addition of membrane come from? One explanation is that the high intracellular hydrostatic pressure triggered the fusion of a large amount of extrasynaptic vesicles to the plasma membrane as previously described during Ca2+ uncaging (Vincent et al., 2014).’8) Results and Discussion, third paragraph: could the authors clarify the reference to 'mechanosensitive' Cav1.3 channels. Is it just that calcium channels are added to the membrane during the massive vesicular fusion caused by increased hydrostatic pressure?

We agree with the reviewer, ‘we cannot exclude the addition of Ca2+ channels to the plasma membrane during the massive vesicular fusion caused by increased hydrostatic pressure. However, this mechanism appears unlikely in regards to results obtained in non-secretory cells such as smooth muscle and HEK cells where similar effect on Ca2+ channels were obtained (Lyford et al., 2002).’ This point is now added in the seventh paragraph of the Results and Discussion section.

9) In the Results and Discussion, third paragraph, please rephrase the following passage: “Notably, a change in Ca2+ current amplitude…”. The authors should replace “change” for “reduction”.

This is now indicated in the seventh paragraph of the Results and Discussion section (‘Notably, an increase in Ca2+ current amplitude in low bath hydrostatic pressure (equivalent to increasing intracellular pressure) was also reported in dissociated guinea pig vestibular hair cells (Duong Dinh et al., 2009; Haasler et al., 2009)’).

10) Results and Discussion, last paragraph: F-actin regulates vesicle trafficking by sensing and regulating focal changes in hydrostatic pressure? What are these 'focal changes in hydrostatic pressure'? How would these be generated?

To clarify this issue, this section has been rewritten to read: ‘In conclusion, we propose that a synaptic F-actin network tightly controls the flow of synaptic vesicles during exocytosis at the IHC ribbons. This synaptic F-actin network also influences the sensitivity of exocytosis to hydrostatic pressure changes, presumably through a regulation of membrane tension at the active zones.’

11) Figure 1: provide hair cell outline in right hand panel at hi mag. Is this a z-axis projection?

Done.

Panel B mislabeled as panel C on figure.

Done.

What are the large black holes at the bottom of latrunculin-treated IHCs?

‘The black holes at the base of the IHCs likely indicated swollen IHC active zones produced by the synaptic F-actin disorganization’. This point is now added in the legend of Figure 1C.

What are the dimensions of F-actin cages in latrunculin?

The size of the synaptic F-actin cages in latrunculin treated IHCs could not be measured since they were completely disorganized (see statement in the first paragraph of the Results and Discussion section).

Are these center-to-center distances of ribbon and CaV1.3? Please specify in the figure legend.

We now specify in the legend of Figure 1D that these distances are the center mass distances.

For correspondence

Competing interests

Funding

Agence Nationale de la Recherche (ANR-10-LABX-65)

Christine Petit

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the Bordeaux Imaging Center for providing cutting-edge instruments for high resolution confocal microscopy and Professor Ray Cooke for copyediting the manuscript. This work was supported by the French state program ‘‘Investissements d’Avenir’’ (ANR-10-LABX-65; to C.P.).

Ethics

Animal experimentation: This study was performed in accordance with the guidelines of the Animal Care Committee of the European Communities Council Directive (86/609/EEC) and were approved by the ethics committee of the University of Bordeaux (animal facility agreement 155 number C33-063-075)

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